Application of HERC5 protein in preparation of anti-PDCoV proliferation drug
By studying the role of pig-derived HERC5 protein, the HERC5 protein expression cell line was constructed, which solved the problem of the unclear relationship between HERC5 and PDCoV proliferation, achieved significant inhibition of PDCoV proliferation, and provided a theoretical basis for the development of anti-PDCoV drugs.
Patent Information
- Application Number
- CN202411468263.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-01-10
AI Technical Summary
Pig delta coronavirus (PDCoV) has caused serious harm to the pig farming industry. The existing technology has not yet clarified the relationship between HERC5 protein and PDCoV proliferation, and there is a lack of effective basis for the development of anti-PDCoV proliferation drugs.
By studying the effect of pig-derived HERC5 protein on PDCoV proliferation, a cell line that stabilizes HERC5 protein expression was constructed. It was found that HERC5 protein has the ability to significantly inhibit PDCoV proliferation, providing a theoretical basis for screening and research and development of new anti-PDCoV drugs.
The constructed HERC5 protein expression cell line significantly inhibited the proliferation of PDCoV, providing a theoretical basis for the development of anti-PDCoV drugs, and demonstrating the application potential of HERC5 protein in preventing and controlling PDCoV.
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Figure CN119280379A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of antiviral drugs, and more specifically to the application of HERC5 protein in the preparation of anti-PDCoV proliferation drugs. Background Art
[0002] Porcine deltacoronavirus (PDCoV) is one of the main pathogens causing diarrhea in pigs, causing serious damage to the pig industry. Interferon-stimulated genes (ISGs) and their expression products can activate the body's antiviral state, inhibit pathogen proliferation, and activate immune cells to regulate immune function. HREC5 is an antiviral immune protein induced by IFN-α / β signal transduction. Several studies have confirmed that HERC5 plays a core role in mammalian innate immunity by disrupting viral replication through ubiquitination of viral proteins. However, the relationship between HERC5 and PDCoV proliferation is still unclear.
[0003] In summary, exploring the effect of porcine HERC5 on the proliferation of PDCoV is an urgent problem that technicians in this field need to solve. Summary of the invention
[0004] In view of this, the present invention provides the use of HERC5 protein in the preparation of anti-PDCoV proliferation drugs. By studying the role of porcine HERC5 in PDCoV proliferation, an application basis is provided for the prevention and control of PDCoV.
[0005] In order to achieve the above object, the present invention adopts the following technical solution:
[0006] Application of HERC5 protein in the preparation of anti-PDCoV proliferation drugs.
[0007] Furthermore, overexpression of HERC5 protein was used to inhibit PDCoV proliferation.
[0008] Furthermore, the HERC5 protein is a porcine HERC5 protein.
[0009] Furthermore, the amino acid sequence of the HERC5 protein is shown in SEQ ID NO.1;
[0010]
[0011] Furthermore, the PDCoV is the PDCoV-TJ1 strain.
[0012] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:
[0013] The present invention constructs a cell line in which a lentivirus stably expresses the HERC5 protein, and finds that the HERC5 protein has a significant inhibitory effect on the proliferation of the PDCoV virus. The present invention provides a theoretical basis for the screening and development of new anti-PDCoV drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0015] Figure 1 The effect of PDCoV on HERC5 expression detected by RT-qPCR in Example 1 of the present invention, wherein the left figure represents 36h, the right figure represents 48h, and *** represents P<0.001 and extremely significant difference;
[0016] Figure 2 The effect of inactivated PDCoV on HERC5 expression detected by RT-qPCR in Example 1 of the present invention, wherein the left figure represents 36h and the right figure represents 48h;
[0017] Figure 3 The inactivation of the virus detected by IFA in Example 1 of the present invention;
[0018] Figure 4 This is a PCR identification diagram of the HREC5 overexpression vector in Example 1 of the present invention;
[0019] Figure 5 The RT-qPCR method in Example 1 of the present invention detects the effect of overexpressing HERC5 at different doses on the proliferation level of PDCoV, wherein the left figure represents 36h, the right figure represents 48h, and *** represents P<0.001 and extremely significant difference;
[0020] Figure 6 The RT-qPCR method in Example 1 of the present invention detects the mRNA level of HERC5 after transfection with different doses of pCAGGS-HERC5, wherein the left figure represents 36h, the right figure represents 48h, and *** represents P<0.001 and extremely significant difference;
[0021] Figure 7 The CCK-8 method for detecting cell viability in Example 1 of the present invention;
[0022] Figure 8 The results of real-time fluorescence quantitative PCR after the HERC5 lentiviral recombinant vector infected IPEC-J2 in Example 1 of the present invention, where *** represents a very significant difference of P < 0.001;
[0023] Fig. 9 This is the effect of stable overexpression of HERC5 on the proliferation level of PDCoV detected by real-time fluorescence quantitative PCR in Example 1 of the present invention, wherein *** represents extremely significant difference of P < 0.001. DETAILED DESCRIPTION
[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0025] The PDCoV strain used in the present invention is the PDCoV-TJ1 strain, which was isolated and preserved by the livestock and poultry disease prevention and control innovation team of the Animal Husbandry and Veterinary Research Institute of Tianjin Academy of Agricultural Sciences; (Zheng Li, Li Xiuli, Yan Minghua * , Ren Weike, Zhang Lei, Lu Chao, Tian Xiangxue, Han Wei. Isolation, identification and biological characteristics analysis of porcine deltacoronavirus TJ1 strain [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine (Chinese Core Journal). 2018, 45(1): 219-224).
[0026] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.
[0027] Example 1
[0028] 1 Materials and methods
[0029] 1.1 Construction and transfection of eukaryotic expression vector
[0030] According to the gene sequence of porcine HERC5 published in NCBI's GenBank, amplification primers were designed (see Table 1). Using IPEC-J2 cell total RNA as a template, a porcine HERC5 fragment with a Myc tag in series at the N'terminus was amplified and connected to the target vector to obtain the porcine HREC5 fusion overexpression vector pCAGGS-HERC5. The cloned vector was sent to Genewise Biotechnology Co., Ltd. for sequencing.
[0031] Table 1 Primer sequences
[0032]
[0033] Pig HERC5 sequence number:
[0034] Sus scrofa HECT and RLD domain-containing E3 ubiquitin protein ligasefamily member 5(HERC5)mRNA,complete cds
[0035] GenBank:OR493513.1
[0036] The amino acid sequence of porcine HERC5 protein is:
[0037]
[0038] IPEC-J2 cells in good growth state were plated in 6-well plates at appropriate ratios and cultured in a 37°C, 5% CO2 incubator for 12 hours until the cell confluence reached 70% to 80%. Using the untransfected wells as controls, the eukaryotic overexpression vector pCAGGS-HERC5 of porcine HERC5 with Myc-tag was transfected into IPEC-J2 cells using the transfection reagent Lipo3000 according to the instructions.
[0039] 1.2 Virus inoculation
[0040] IPEC-J2 cells were seeded in 6-well plates and complete culture medium was added. When the cell confluence reached about 80%, the culture medium was discarded and the cells were washed three times with PBS. Pancreatin was added to serum-free DMEM culture medium to prepare a virus dilution solution with a final pancreatin concentration of 10 μg / mL. The virus stock solution was diluted and inoculated according to the multiplicity of infection (MOI) of 0.1 PDCOV per well of cells, and incubated for 4 hours after virus inoculation. The virus dilution solution containing PDCOV was discarded and the maintenance solution (serum-free culture medium containing 6 μg / mL pancreatin) was added.
[0041] 1.3 Inoculation of inactivated virus
[0042] Inactivate the virus stock solution under ultraviolet light for 30 minutes and inoculate in the same way as in 1.2.
[0043] 1.4 Indirect immunofluorescence assay (IFA) to detect virus inactivation
[0044] Using uninfected IPEC-J2 cells as controls, IPEC-J2 cells infected with PDCoV for 24h at 0.1MOI were washed three times with PBS. Fixed with 4% paraformaldehyde for 30min. The primary antibody against PDCoV S protein was diluted with TBST at a dilution ratio of 1:100, and the goat anti-rabbit secondary antibody was diluted at a dilution ratio of 1:200. After washing the fixed cells three times with PBS, the primary antibody was incubated at 37℃ for 1h. Washed with PBS three times, and the secondary antibody was incubated at 37℃ for 1h. After washing with PBS three times, the cells were stained with DAPI nucleic acid dye for 10min. After washing the staining solution with PBS, the IFA staining results were observed under a fluorescence microscope.
[0045] 1.5 Total RNA extraction, reverse transcription and real-time fluorescence quantitative PCR detection of target gene mRNA levels
[0046] Total RNA from cells was extracted according to the instructions of the Trizol kit and stored in a refrigerator (-80°C). The extracted RNA samples were reverse transcribed into cDNA using a reverse transcription kit. Real-time fluorescence quantitative PCR was performed using SYBR enzyme from Genewise according to the manufacturer's protocol. The primer pairs used are shown in the following table.
[0047] Table 2 Quantitative primer sequences
[0048]
[0049] The relative expression changes of HERC5 and PDCoV M genes were analyzed by normalization using the 2-ΔΔCt method with GAPDH as the internal reference. All analyses were performed with three technical and biological replicates.
[0050] 2 Results
[0051] Effect of PDCoV infection on HERC5 expression
[0052] IPEC-J2 cells were incubated with PDCoV, and the cells were harvested at 36h and 48h after infection to extract total RNA. RT-qPCR was used to detect the transcription level of HERC5. Figure 1 As shown, the mRNA level of HERC5 was significantly downregulated at 36h and 48h after PDCoV inoculation.
[0053] To determine whether the downregulation of HERC5 is dependent on viral replication, PDCoV was inactivated by ultraviolet irradiation, and the inactivated virus was inoculated into IPEC-J2 cells, and indirect immunofluorescence was performed at 24 h. The results showed that the virus was completely inactivated ( Figure 3 ). After inactivated virus infection of IPEC-J2 cells, PDCoV inactivation failed to induce downregulation of HERC5 expression ( Figure 2 ), indicating that viral infection-induced downregulation of HERC5 expression is dependent on PDCoV replication.
[0054] 2.2 The inhibitory effect of HREC5 overexpression on PDCoV proliferation is dose-dependent
[0055] Construction of HERC5 overexpression vector pCAGGS-HERC5 (PCR identification results see Figure 4 Different doses (0.5, 1, 2 μg / L) of pCAGGS-HERC5 and empty vector pCAGGS were transiently transfected into IPEC-J2 cells for 36 h and 48 h, respectively, to observe the role of HERC5 in PDCoV infection. RT-qPCR was used to detect the mRNA levels of PDCoV mRNA and HERC5.
[0056] like Figure 5 , Figure 6As shown, compared with the mRNA level of PDCoV in IPEC-J2 cells transfected with empty vector, the mRNA level of HERC5 in IPEC-J2 cells transfected with pCAGGS-HERC5 was significantly upregulated, while the mRNA level of PDCoV was significantly inhibited at 36h and 48h after infection in a dose-dependent manner. These data indicate that overexpression of HERC5 protein inhibits the replication of PDCoV in IPEC-J2 cells.
[0057] 2.3 Cell lines stably overexpressing HERC5 inhibit the proliferation of PDCoV on IPEC-J2 cells
[0058] Using IPEC-J2 cell total RNA as a template, the obtained porcine HERC5 fragment was amplified (primer sequence is shown in the table below), and connected to the target vector with tandem 6×His and Flag tags to obtain the porcine HERC5 double-tag fusion overexpression lentiviral vector CMV-HERC5-His-FLag. After restriction digestion and identification, the cloned vector was sent to Genewise Biotechnology Co., Ltd. for sequencing.
[0059] Table 3 Primer sequences
[0060]
[0061] The IPEC-J2 cell line (CMV-HERC5) stably overexpressing HERC5 was established by lentiviral packaging technology. For details on lentiviral packaging and infection technology, please refer to "Novel interacting proteins identified by tandem affinity purification and mass spectrometry associated with IFITM3 protein during PDCoV infection." The results of the plasmid concentration measurement used for lentiviral packaging are as follows:
[0062] Table 4 Results of determination of plasmid concentration in lentiviral system
[0063]
[0064] Note: When the OD260 / OD280 value is between 1.8 and 2.0 and the concentration is not less than 300 ng / μL, it meets the experimental requirements.
[0065] IPEC-J2 cells stably transfected with empty plasmid CMV by lentivirus were used as negative control (CMV). CCK-8 assay showed no significant change in cell proliferation activity ( Figure 7). HERC5 overexpression in IPEC-J2 cells was detected by RT-qPCR. HERC5 mRNA levels were significantly increased in CMV-HERC5 compared with CMV and control IPEC-J2 cells ( Figure 8 ), indicating that the lentiviral cell line stably expressing the target gene was successfully constructed.
[0066] IPEC-J2 cells overexpressing HERC5 were infected with PDCoV virus, and IPEC-J2 cells transfected with CMV blank vector were used as the control group. Total RNA of cells was collected at 36h and 48h, and RT-qPCR was used to detect the mRNA level of PDCoV. The results showed that compared with the control group, stable overexpression of HERC5 could significantly inhibit the mRNA level of PDCoV ( Fig. 9 ).
[0067] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0068] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. Application of HERC5 protein in the preparation of anti-PDCoV proliferation drugs.
2. Application as claimed in claim 1, It is characterized by, Overexpression of HERC5 protein is used to inhibit PDCoV proliferation.
3. Application as claimed in claim 1, It is characterized by, The HERC5 protein is a porcine HERC5 protein.
4. Application as claimed in claim 1, It is characterized by, The amino acid sequence of the HERC5 protein is shown in SEQ ID NO.1; MERRPRRRSRGGARSAPGRGPAAGPPRSPRGAKLWLFPSAPGLRSALAGRTEATRQMCCTGGRLAVLERGGAGVEVHQLPAGSDGARKPKCIKLGKKMKIHSMDQGAEHMLILSSDGKPFEYNYSIEH ARFQCILQEKIIIQITCGDYHSLALSKGGELFAWGENLHGQLGVGRGIASTPTPQMVEHLYGVPLAQVSAGGAHSVALSMSGNVYSWGGNDFGQLGLGHTDDADFPSRIEALDGQHVEFLACGASHTAL LTKGGLVLTFGDGRCGQLGHNSTQNELRPRLVTELVGKRVTQMACGREHTLAYVSDLGKVFAFGSGTEGQLGNGGTHNQLIPRPMKLPSNEELKFESRTSEKELIMIAGGNQSILLWMEKENSYVNLR RKIPTLNEGTVKRWLADVGTKQWQNTKREIREIFSSPACLTGSLFLRERITGGRTSVHLDLNTARTALSELARKDCITNTITACLRENLLKALPCHSPHREAVEVFFLLPECPVMHDDNNWESLVVPFAE AVGKMSDPSSGVLEEYWASLGESAFIRLVQMFKRAIVAQLRCWTKSAESNCHIKALLELLKKLHRVNQTKCHLPESIFTVNELTDYLDFHEDAYRRSFWKVSSDTSVDIQYPVIFSHPFIFNILSKI KLLYADSLLKIQQKKIRAYMPLAGILGQEGSELALPPTLNLLVRRGHLIEDVFSHLSRYENEDLRRELLVSFSGEIGYDFGGVRAEFFHCLFEELTQPEYGLFTYPEDASCMWFPARPKFENKSYFFFG ILCGLSLFNCNVANIPFPLALFKKLLDQEPSLEDLKELSPVLGQSLQTLLDYEDDDFGKIFHVNMHWDKDSVDLIPHGSDVIVDQSNKREYISKYVDYIFNISVKEVYEEFQRGFYRVCDKEIIEF FHPEELKDVIIGNTDYDWETFEKNARYEHGYDNSHPTIVMFWKALHKLTLEEKKKFLVFLTGTDRIQVRGLKNMKITFCCPENMNEKDPIRAQTCFSVLYLPKYSTMERVEEALQVAISNHRGFG, SEQ ID NO.
1.
5. Application as claimed in claim 1, It is characterized by, The PDCoV is PDCoV-TJ 1 strains.